DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Application and Claim Status Summary
Claims 1- 32 are pending.
Claims 1- 14, 16- 29, 31, and 32 are considered on the merits.
Claims 2, 15, 17, 30 are objected.
Claims 1- 14, 16, 31, and 32 are rejected.
Claims 17- 29 are allowed.
This action is written in response to applicant's correspondence received 2024 March 25. Claims 1- 32 are currently pending, and Claims 1- 14, 16- 29, 31, and 32 are examined on the merits.
Claim Objections
Claims 2 and 18 are objected to because of the following informalities: claims 2 and 18 recite “the first RNA molecule comprises a spacer specific for the first excision site“ and “the second RNA molecule comprises a spacer specific for the second excision site.” However, claim 1 (and claim 17), recites in clause d) “a first RNA molecule comprising a sequence specific for the first excision site” and “a second RNA molecule comprising a sequence specific for the second excision site.” It is this examiner’s interpretation that the “spacers” recited in claims 2 and 18 are the same as the respective “sequences” referenced in claim 1 (and claim 17). The use of alternate language for the same structure from claim to claim is not a best practice (see MPEP 21703). Therefore, the claim should be modified to promote best practice.
Claim 17 is objected to because of the following informalities: the claim has two clauses labeled “c).” This is a grammatical error. Each clause labeled with a sequential letter as an identifying mark should receive its own letter.
Appropriate correction is required.
The following is a quotation of 35 U.S.C. 112(e):
(e) REFERENCE IN MULTIPLE DEPENDENT FORM. — A claim in multiple dependent form shall contain a reference, in the alternative only, to more than one claim previously set forth and then specify a further limitation of the subject matter claimed. A multiple dependent claim shall not serve as a basis for any other multiple dependent claim. A multiple dependent claim shall be construed to incorporate by reference all the limitations of the particular claim in relation to which it is being considered.
Claims 15 and 30 are objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n). Accordingly, the claims have not been further treated on the merits.
Claim 15 dependencies refer to claim 1 and claim 12.
Claim 30 dependencies refer to claim 1, claim 12 and claim 17.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 5, 6, 21, and 22 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Those claims included in the statement of rejection but not otherwise discussed are rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
Claim 5 depends from claim 1 and claim 6 depends from claim 5. Claim 1 recites, “said episomal replicon comprising a backbone sequence and a donor nucleic acid sequence.” This clause is interpreted to mean these are two distinct and discrete sequences. Claim 1 then describes the composition of the donor nucleic acid, “wherein said donor nucleic acid sequence comprises in order: 5' - homologous recombination sequence 1 - sequence of interest - homologous recombination sequence 2 - 3';” and further recites “wherein the backbone sequence comprises a first excision site positioned adjacent to homologous recombination sequence 1 and a second excision site positioned adjacent to homologous recombination sequence 2.”
However, Claim 5 recites “wherein each terminus of the excised nucleic acid comprises nucleic acid sequence derived from the backbone sequence” and claim 6 recites “wherein the excised donor nucleic acid comprises 6 or fewer base pairs of nucleic acid sequence derived from the backbone sequence at each terminus.” Based on the structure claimed in claim 1, it is unclear how any part of the backbone derived sequence may remain as part of the excised nucleic acid when the excision point is adjacent to the terminal ends of the donor nucleic acid. As described, in claim 1, the excision point would create a cut separating all of the backbone from all of the donor. This interpretation is further supported by the instant specifications, “The excision sites are adjacent to the homologous recombination sequences of the donor nucleic acid. Preferably each excision site is contiguous with the homologous recombination sequence. In such embodiments, there are no base pairs in between the sequences required for excision and the homologous recombination sequence.” [Page 16, lines 26- 29].
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 6 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 6 depends from claim 5 which depends from claim 1. As described above, claim 1 describes an excised donor nucleic acid that terminates with the regions of homology on either end, and no bases derived from the backbone are included. Therefore, the current limitation “wherein the excised donor nucleic acid comprises 6 or fewer base pairs of nucleic acid sequence derived from the backbone sequence at each terminus” does not further limit the claim.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1- 3, 7, 9- 13 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wang (Wang et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016)).
Regarding claim 1, Wang’s Figure 1 (shown below) provides visual guidance in mapping Wang’s teachings to the instant claim. The figure depicts an E. coli cell at different stages of the method. Inside the cell, is depicted the BAC DNA (episomal replicon) with the synthetic DNA (sequence of interest) and just below is the E. coli chromosome (target nucleic acid/genome).
Wang teaches the preamble of the claim (method of introducing a sequence of interest into a target nucleic acid), “Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA…” (Abstract). Furthermore, the context of Wang’s statement above, and Figure 1a below depicts all of the elements of clause a), arranged as recited. Wang’s teachings or clarifications thereof are shown in brackets, mapped adjacent to the claim limitation. Clause a) recites,
“a) providing a host cell [described above]
said host cell comprising an episomal replicon, [described above]
said episomal replicon comprising a backbone sequence [the backbone sequence is the BAC excluding the HR1, s. DNA, -2 sequence, +2 sequence, and HR2 sequences; notice the backbone includes the excision site sequences noted as deep blue and orange rectangles] and a donor nucleic acid sequence [the donor nucleic acid sequence is the HR1, s. DNA, -2 sequence, +2 sequence, and HR2 sequences of the BAC],
wherein said donor nucleic acid sequence comprises in order: 5' - homologous recombination sequence 1 [BAC HR1 sequence]- sequence of interest [BAC s. DNA sequence]- homologous recombination sequence 2 - 3' [BAC HR2 sequence],
wherein the backbone sequence comprises a first excision site positioned adjacent to homologous recombination sequence 1 [the excision site sequence is indicated by the deep blue rectangle and its respective excision site is indicated by the deep blue triangle] and a second excision site positioned adjacent to homologous recombination sequence 2 [the excision site sequence is indicated by the orange rectangle and its respective excision site is indicated by the orange triangle],
said host cell further comprising a target nucleic acid [described above];”
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Wang continues their statement from above, “… through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection” (Abstract). Here, “lambda-red-mediated recombination” reads on clause b). Wang’s system utilizes in vivo components (helper proteins) of the lambda-red-mediated recombination to combine the donor nucleic acid with the target nucleic acid. Similarly, “in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9” reads on clauses c), d), and e).
Regarding clause c), just below the first E. coli pictograph, is noted the blue and orange excision site triangles described above and the text “excision of dsDNA by CRISPR/Cas9.” This note indicates all of the components and methods of a CRISPR/Cas9 system were employed in vivo, including target DNA cleavage sites encoded and marked by PAMs, gRNAs (including target specific guide/spacer RNAs and scaffold RNAs), Cas9 nuclease, and assembly of the ribonucleoprotein complex.
Regarding clause d), the “first RNA molecule comprising a sequence specific for the first excision site” and “second RNA molecule comprising a sequence specific for the second excision site” is described above by the blue triangle/gRNA and the orange triangle/gRNA respectively. Similarly, the limitation “wherein the first and the second RNA molecules contribute to directing the RNA-guided DNA endonuclease during excision” describes the mechanism/function of the CRISPR/Cas9 system including the two gRNAs (deep blue and orange) described above.
Regarding clause e), similarly as described above, the limitation “inducing excision of said donor nucleic acid sequence by the RNA-guided DNA endonuclease” describes the mechanism/function of the CRISPR/Cas9 system including the two gRNAs (deep blue and orange).
Furthermore, “through the in vivo excision … coupled to lambda-red-mediated recombination …” reads on clause “f) incubating to allow recombination between the excised donor nucleic acid and said target nucleic acid.” By allowing the cells to incubate, the lambda-red recombination components described above will assemble the donor and target nucleic acids at the donor’s excision points.
Regarding claim 2, 3, Wang teaches their endonuclease is a CRISPR-Cas9 nuclease as described above and the first and second spacers are specific to their respective excision sites.
Regarding claim 7, Wang teaches their episomal replicon is a bacterial artificial chromosome (BAC) as described above.
Regarding claim 9, Wang teaches the target nucleic acid is the genome of the host cell as described above.
Regarding claims 10 and 11, Wang teaches the host cell is an E. coli prokaryotic cell as described above.
Regarding claims 12 and 13, Wang teaches all of the elements of claim 1. Furthermore, claim 12 is simply a single iteration of claim 1 and claim 13 allows multiple iterations, which Wang teaches “Iteratively replacing large sections of the genome with synthetic DNA via REXER will enable genome stepwise interchange synthesis (GENESIS) for whole-genome replacement (Fig. 2a).” (page 60, 2nd column, 6thparagraph).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 4 and 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016)) as applied to claim 1 above, and further in view of Xie (Xie et al. Sci Rep. 2017 May 24;7(1):2320.).
Regarding claim 4, Wang teaches claim 1 as described above.
Wang does not teach “wherein the first RNA molecule and/or the second RNA molecule are encoded by the episomal replicon.”
Xie teaches they are in the same field of endeavor, genetic manipulation, “Here, we developed an episomal vector based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs.” (Abstract). Xie’s Figure 1a (below left) shows a map of the episomal replicon encoding the spacer (gRNA) of the instant claim and Figure 3a (below right) shows a map of the episomal replicon encoding two spacers (gRNAs).
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Xie describes the figure in the legend “Expression of two gRNAs on the epiCRISPR vector for double-gene knockout.” Furthermore, Xie teaches their method utilizes the same endogenous homologous repair system as Wang, “The DSBs can also be repaired by homology-directed repair (HDR) … leading to knock-in of precise mutations or reporters” (page 1, 2nd paragraph). Furthermore, Xie describes their motivation for developing their technique is “high efficiency (8–76%) of genome editing by using an episomal vector to express Cas9 and gRNA” (page 2, 2nd paragraph). Xie further encourages others to use their own technique boasting, “The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates” (Abstract).
It would have been obvious for a person having ordinary skill in the art (PHOSITA) before the effective filing date of the claimed invention to have encoded the first and second RNAs into the BAC as taught by Xie with the method of Wang because Xie provided the motivation “high efficiency (8–76%) of genome editing by using an episomal vector to express Cas9 and gRNA” and, “The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates.” Xie’s system was successful in some instances as much as 100%. Therefore, a PHOSITA would reasonably expect similar success by following Xie’s suggestion to encode the RNA into the episomal replicon method of Wang.
Regarding claim 16, Wang teaches claim 1 and 12 as described above.
Furthermore, Wang and Xie together teaches the first RNA molecule and/or the second RNA molecule are encoded by the episomal replicon as described above. Therefore, as described above, Wang and Wang-Xie teach the instant limitation, “wherein part (i) comprises the use of a donor-nucleic-acid-sequence-encoding episomal replicon comprising a first backbone sequence, and part (ii) comprises the use of a donor-nucleic-acid-sequence-encoding episomal replicon comprising a second backbone sequence, wherein…encodes the first RNA molecule specific for the first excision site within said first backbone sequence, and encodes the second RNA molecule specific for the second excision site within said first backbone sequence… and … encodes the first RNA molecule specific for the first excision site within said second backbone sequence, and encodes the second RNA molecule specific for the second excision sites within said second backbone sequence.”
As described below, Wang further teaches their “the first backbone sequence comprises a first marker or set of markers” and “the second backbone sequence comprises a second marker or set of markers.”
Wang describes the selection markers labeled in the figure above “+1,” “-1,” “+2,” and “-2” with the following statement, “…. +1 is KanR [kanamycin;], −1 is rpsL, +2 is CmR, −2 is sacB.” (Figure 1 legend). Their Figure 2a (below) provides additional clarity.
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Wang explains in further detail, “We created E. coli MDS42rpsLK43R/rK (a genome-minimized strain of E. coli in which the genomic copy of rpsL contains a K43R mutation conferring resistance to streptomycin, and the −1/+1 selection cassette encoding an rpsL–KanR fusion inserted between cra and mraZ) containing a bacterial artificial chromosome (BAC) in which the −2/+2 cassette (encoding sacB–CmR) is flanked by the HR1 and HR2 sequences” (page 60, 1st column, 1st paragraph).
Claim(s) 8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016)) as applied to claim 1 above, and further in view of Fredens (Fredens et al. Total synthesis of Escherichia coli with a recoded genome. Nature. 2019 May;569(7757):514-518.).
Regarding claim 8, Wang teaches all of the elements of claim 1. Wang further teaches transforming their BAC into E. coli via “We transformed each BAC into E. coli MDS42rpsLK43R/rK” (page 60, 2nd column, 3rd paragraph). Wang further teaches “We named our approach REXER 2 (replicon excision for enhanced genome engineering through programmed recombination” (page 60, 2nd column, 3rd paragraph).
Wang does not teach “wherein the episomal replicon is delivered to the host cell by conjugative transfer.”
Fredens teaches a bacterial recombineering using similar REXER methods as Wang “We assembled bacterial artificial chromosomes (BACs) for REXER” (page 515, 1st column, 3rd paragraph and see Freden’s Extended Data Fig. 1a below). Furthermore, Fredens emphasizes the similarity of methods “REXER uses CRISPR/Cas9- and lambda-red-mediated recombination to replace genomic DNA with synthetic DNA provided from an episome (BAC)” (Extended Data Fig. 1 legend).
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Fredens teaches their method of introducing the BAC into bacteria via conjugative transfer “We developed a conjugation-based strategy to assemble the recoded sections into a single genome” (page 516, 2nd column, 3rd paragraph).
It would have been obvious for a person having ordinary skill in the art (PHOSITA) before the effective filing date of the claimed invention to have used the conjugative transfer method of Fredens with the method of introducing a sequence of interest into a target nucleic acid of Wang because Wang suggested using alternative BAC transfer methods. Wang suggests investigating alternative methods of BAC delivery by noting their challenges “The overall efficiency [of lambda-red-mediated recombination] decreases markedly with increasing dsDNA length, and we hypothesized that this decrease results primarily from challenges in efficiently delivering intact dsDNA into cells” (page 59, 2nd column 3rd paragraph). A PHOSITA would have seen Fredens conjugative transfer using Wang’s method of introducing a sequence of interest into a target nucleic acid and reasonably expected similar success.
Claim(s) 31 and 32 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang (Wang et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016)) as applied to claim 1 above, and further in view of Narayanan (Narayanan K, Chen Q. Bacterial artificial chromosome mutagenesis using recombineering. J Biomed Biotechnol. 2011;2011:971296.).
Regarding claims 31 and 32, Wang teaches all of the elements of claim 1.
Wang further teaches transforming their BAC into E. coli via “We transformed each BAC into E. coli MDS42rpsLK43R/rK” (page 60, 2nd column, 3rd paragraph). Wang further teaches “We named our approach REXER 2 (replicon excision for enhanced genome engineering through programmed recombination” (page 60, 2nd column, 3rd paragraph).
Wang does not teach “wherein the host cell is lacking competent recA and/or recO.”
However, recall Wang teaches the use of the lambda-red recombination system, “… through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection” (Abstract).
Narayanan teaches “Lamda Red pathway” recombination as a “BAC Modification Strategies” in their Table 1 and further teaches, “Early research that led to the development of recombineering (Figure 2) can be traced back to the exploitation of E. coli mutant strains that carry functional phage recombination mechanisms derived from lambda … genes…” (page 5, 2nd column, 2nd paragraph). Furthermore, Narayanan teaches the benefits (motivation) of recA deficient methods, “The key feature of recombineering strategies is that it is independent of E. coli endogenous homologous recombination functions, and it therefore, can be transferred using mobile plasmids into hosts that are recombination deficient (e.g., RecA background) to introduce recombination proficiency transiently” (page 2, 1st column, 3rd paragraph).
It would have been obvious for a person having ordinary skill in the art (PHOSITA) before the effective filing date of the claimed invention to have used recA deficient cells as taught by Narayanan with the method of introducing a sequence of interest into a target nucleic acid as taught by Wang because Narayanan provided the motivation to do so. As noted above, Narayanan highlighted the features of recombineering systems that exploit lambda-red and are recA deficient. Therefore, a PHOSITA would follow Narayanan’s suggestion to us host cells that lacked competent recA with Wang’s method.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1- 3, 7, and 9- 13 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1- 23 of U.S. Patent No. 11667933. Although the claims at issue are not identical, they are not patentably distinct from each other because of the following reasons.
Regarding instant claim 1, Claim 1 of the reference patent recites “A method comprising (a) providing a host cell said host cell comprising an episomal replicon, said episomal replicon comprising a donor nucleic acid sequence, said host cell further comprising a target nucleic acid…” Here, the reference claim reads on the instant claim 1 recitation “A method of introducing a sequence of interest into a target nucleic acid, the method comprising a) providing a host cell said host cell comprising an episomal replicon… said host cell further comprising a target nucleic acid”
The reference claim 1 further recites, “(b) providing helper protein(s) capable of supporting nucleic acid recombination in said host cell….” Here, the reference claim reads on the instant claim 1 recitation “b) providing helper protein(s) capable of supporting nucleic acid recombination in said host cell.”
The reference claim 1 further recites, “(c) providing helper protein(s) and/or RNAs capable of supporting nucleic acid excision in said host cell ….” Here, the reference claim reads on the instant claim 1 recitation “c) providing an … DNA endonuclease.”
The reference claim 1 further recites, “wherein said donor nucleic acid sequence comprises in order: 5′-homologous recombination sequence 1-sequence of interest-homologous recombination sequence 2-3′ wherein said sequence of interest comprises both a positive selectable marker and a first negative selectable marker…” Here, the reference claim reads on the instant claim 1 recitation “said episomal replicon comprising … and a donor nucleic acid sequence, wherein said donor nucleic acid sequence comprises in order: 5′—homologous recombination sequence 1—sequence of interest—homologous recombination sequence 2—3′.”
The reference claim 1 further recites, “(d) inducing excision of said donor nucleic acid sequence…” Here, the reference claim reads on the instant claim 1 recitation “e) inducing excision of said donor nucleic acid sequence by the … DNA endonuclease.”
The reference claim 1 further recites, “(e) incubating to allow recombination between the excised donor nucleic acid and said target nucleic acid…” Here, the reference claim reads on the instant claim 1 recitation “f) incubating to allow recombination between the excised donor nucleic acid and said target nucleic acid.”
The reference claim 8 recites, “wherein said excised donor nucleic acid begins with said homologous recombination sequence 1 and ends with said homologous recombination sequence 2.” The reference claim 11 recites, “wherein said episomal replicon comprises in order: excision cut site 1-donor nucleic acid sequence-excision cut site 2.” Together, the reference claims describe the structure that reads on the instant claim 1 recitation “said episomal replicon comprising a backbone sequence… wherein the backbone sequence comprises a first excision site positioned adjacent to homologous recombination sequence 1 and a second excision site positioned adjacent to homologous recombination sequence 2.” Furthermore, this reading describes a sequence specific excision point, and therefore claims 8 and 11, in combination with the earlier recitation of the reference claim “(c) providing helper protein(s) and/or RNAs capable of supporting nucleic acid excision” reads on the following instant claim 1 recitation “providing an RNA-guided DNA endonuclease… inducing excision of said donor nucleic acid sequence by the RNA-guided DNA endonuclease” and furthermore reads on the following portion of clause d), “d) providing a first RNA molecule comprising a sequence specific for the first excision site and a second RNA molecule comprising a sequence specific for the second excision site, wherein the first and the second RNA molecules contribute to directing the RNA-guided DNA endonuclease during excision.”
Regarding instant claims 2 and 3, Claim 21 of the reference patent recites “wherein said helper protein(s) and/or RNAs capable of supporting nucleic acid excision comprise CRISPR/Cas9 proteins/RNAs.” Here, the reference claim reads on the instant claim 2 recitation “wherein the RNA-guided DNA endonuclease is a CRISPR-Cas nuclease, the first RNA molecule comprises a spacer specific for the first excision site, and the second RNA molecule comprises a spacer specific for the second excision site;” and instant claim 3 “wherein the CRISPR-Cas nuclease is Cas9.”
Regarding instant claim 7, Claim 15 of the reference patent recites “wherein at least one of said episomal replicon and said target nucleic acid is a bacterial artificial chromosome (BAC).”
Regarding instant claim 9, Claim 17 of the reference patent recites “wherein said target nucleic acid is the host cell genome.”
Regarding instant claim 10- 13, the SUMMARY OF TIE INVENTION of the reference patent recites, “Here we report a method for single step insertion of 90 kb of synthetic DNA into the E. coli genome, and single step replacement of 100 kb of the E. coli genome with synthetic DNA…” [column 3, lines 15- 19]. Here, the reference reads on instant claims 10 and 11. The reference continues in the same line,”… and demonstrate that the method may be iterated” [column 3, line 19]. Here, the reference reads on instant claims 12 and 13.
Claims 31, 32 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1- 23 of U.S. Patent No. 11667933 as described above in view of Narayanan (Narayanan K, Chen Q. Bacterial artificial chromosome mutagenesis using recombineering. J Biomed Biotechnol. 2011;2011:971296.).
Regarding claims 31 and 32, U.S. Patent No. 11667933 teaches claim 1 as described above.
11667933 does not teach “wherein the host cell is lacking competent recA and/or recO.”
However, recall 11667933 teaches the use of the lambda-red recombination system in claim 22, “The method according to claim 1, wherein said helper protein(s) capable of supporting nucleic acid recombination comprise lambda Red proteins” (Abstract).
Narayanan teaches “Lamda Red pathway” recombination as a “BAC Modification Strategies” in their Table 1 and further teaches, “Early research that led to the development of recombineering (Figure 2) can be traced back to the exploitation of E. coli mutant strains that carry functional phage recombination mechanisms derived from lambda … genes…” (page 5, 2nd column, 2nd paragraph). Furthermore, Narayanan teaches the benefits (motivation) of recA deficient methods, “The key feature of recombineering strategies is that it is independent of E. coli endogenous homologous recombination functions, and it therefore, can be transferred using mobile plasmids into hosts that are recombination deficient (e.g., RecA background) to introduce recombination proficiency transiently” (page 2, 1st column, 3rd paragraph).
It would have been obvious for a person having ordinary skill in the art (PHOSITA) before the effective filing date of the claimed invention to have used recA deficient cells as taught by Narayanan with the method of introducing a sequence of interest into a target nucleic acid as taught by 11667933 because Narayanan provided the motivation to do so. As noted above, Narayanan highlighted the features of recombineering systems that exploit lambda-red and are recA deficient. Therefore, a PHOSITA would follow Narayanan’s suggestion to us host cells that lacked competent recA with 11667933’s method.
Allowable Subject Matter
Claims 14, and 15 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claims 14 and 15 share similar claim language that distinguishes the claims over prior art. Claim 14 depends from claim 13, which depends from claim 12, which depends from claim 1. Wang anticipates claims 1, 12, and 13 as described above. However, this examiner was unable to find prior art against the following limitations of claims 14:
the sequence of the first RNA molecule for part (i) is the same for each iteration and/or the sequence of the second RNA molecule for part (i) is the same for each iteration; and the sequence of the first RNA molecule for part (ii) is the same for each iteration and/or the sequence of the second RNA molecule for part (ii) is the same for each iteration.
Claim 15, depends from claim 12, which depends from claim 1. Wang anticipates claims 1 and 12 as described above. Wang similarly teaches the following elements of claim 15 (as described above), “(iii) performing the steps of claim 1 to introduce a third donor nucleic acid sequence into the third target nucleic acid in order to create a fourth target nucleic acid; iterating parts (i), (ii), and (iii),”
However, similar to claim 14, this examiner was unable to find prior art against the following limitations of claim 15: “and wherein the sequence of the first RNA molecule for part (iii) is the same for each iteration and/or the sequence of the second RNA molecule for part (iii) is the same for each iteration.”
For both claims 14 and 15, Wang does not teach using the same first and second RNA [gRNA] molecule for each iteration. Furthermore, this examiner was unable to find similar art using the same gRNA for iterative rounds of homology directed repair recombineering such that claims 14 and 15 would be obvious.
Claims 17- 29 are allowed.
Regarding claim 17, the claim has two clauses labeled “c)” as described above. For purposes of clarity, the first clause “c) providing helper protein(s)…” will be referred to as “c)” and the second clause “c) providing an RNA-guided…” will be referred to as “c)’.”
The closest prior art to the instant claim found by this examiner is Wang (Wang et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016)). Figure 1 (shown below) provides visual guidance in mapping Wang’s teachings to the instant claim. The figure depicts an E. coli cell at different stages of the method. Inside the cell, is depicted the BAC DNA (donor episomal replicon) with the synthetic DNA (donor nucleic acid DNAn) and just below is the E. coli chromosome (assembly episomal replicon).
Wang teaches the preamble of the claim (method for constructing an episomal replicon), “Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA…” (Abstract). Furthermore, within the context of Wang’s statement above, a person having ordinary skill in the art (PHOSITA) will readily see that Figure 1 below depicts the following elements of clause a), arranged as recited:
providing a donor episomal replicon, said replicon comprising:
a backbone, said backbone comprising …, a first homology region HRn … and a second, …, homology region uHR, a first excision site positioned adjacent to HRn and a second excision site positioned adjacent to uHR;
a donor nucleic acid DNAn;
a double selection cassette, comprising positive and negative selection markers.
For additional clarity, Wang describes the CRISPR/Cas9 excision site triangles and selection markers labeled “+1,” “-1,” “+2,” and “-2.” With the following statement, Wang’s teaching reads on clause b), “Triangles indicate spacer RNAs that program cleavage within colour-matched protospacers. REXER 4 augments REXER 2 by adding two extra protospacers (purple and red rectangles) and triggering cleavage with four spacer RNAs. +1 is KanR, −1 is rpsL, +2 is CmR, −2 is sacB.” (Figure 1 legend).
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Wang continues their initial statement from above, “… through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection” (Abstract). Here, “lambda-red-mediated recombination” reads on clause c). Similarly, “in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9” reads on clauses c)’, d), and e). Furthermore, “through the in vivo excision … coupled to … recombination” reads on clause f).
Wang does not teach “said backbone [of the donor episomal replicon] comprising universal spacer sequences, … an integration step n, specific first homology region HRn, and a second, universal, homology region uHR, …” This examiner was unable to find prior art mapping to this claim limitation as it relates to bacterial recombineering. Furthermore, this examiner was unable to find prior art such that this limitation is obvious.
Regarding claims 18- 29, these claims depend directly or indirectly from claim 17.
Conclusion
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/AARON DUREL WARD/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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